OMB No. 0925-0001 and 0925-0002 (Rev. 09/17 Approved Through 03/31/2020) PROJECT SUMMARY Myxopyronins are ?-pyrone antibiotics that function by inhibiting bacterial RNA polymerase through a binding site and mechanism different from those of current antibacterial drugs. In preliminary work, we have performed mode-of-action characterization, lead validation, and initial lead optimization--synthesizing and evaluating more than novel 700 analogs--on myxopyronins. We have identified an exceptionally promising advanced lead: aryl myxopyronin APY281. APY281 exhibits direct activity against drug-sensitive and drug-resistant Gram-positive bacterial pathogens in vitro, potentiated activity against drug-sensitive and drug-resistant Gram-negative bacterial pathogens in vitro, excellent physical properties, excellent intravenous and oral pharmacokinetics in mice, excellent tolerability in mice, potent direct activity against methicillin-resistant Staphylococcus aureus (MRSA) in mice (ED50 = 10 mg/kg iv or po), and potent direct activity against Mycobacterium tuberculosis in mice (ED2log = 200 mg/kg po qd). We also have identified an early lead having a structurally related, but simpler, chemical scaffold: arytalkylcarboxamido phloroglucinol ACP1. ACP1 has the same mode of action, a similar antibacterial profile--including excellent activity against MRSA in mice (ED50 = 2.5 mg/kg iv)--a simpler synthesis, a narrower resistance spectrum, and a lower resistance rate. However, ACP1 has low aqueous solubility. We propose to leverage the crystal structures, structure-activity relationships, and synthetic procedures of our preliminary work in order to design, synthesize, and evaluate improved APY281 analogs having higher efficacy and improved ACP1 analogs having higher efficacy and higher aqueous solubility. In addition, we propose to perform advanced preclinical studies on APY281 and on selected improved APY281 and ACP1 analogs. The primary target pathogens will be drug-resistant and multi-drug-resistant Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. The results will enable nomination of at least one development candidate for a first-in-class, systemically active, orally available, therapeutic agent effective against a broad spectrum of drug-sensitive, drug-resistant, and multi-drug-resistant bacterial pathogens.